Culture of MSC from amniotic fluid
1. Twenty amniotic fluid samples (20 ml) were obtained by amniocentesis performed between 16 and 20 weeks of gestation for fetal karyotyping.
2. A novel two‐stage culture protocol for isolating MSCs was from amniotic fluid.
3. For culturing amniocytes (first stage), four primary in situ cultures were set up in 35 mm tissue culture‐grade dishes using Chang medium (Irvine Scientific, Santa Ana, CA).
4. Microscopic analysis of Giemsa‐stained chromosome banding was performed, and the rules for metaphase selection and colony definition were based on the basic requirements for prenatal cytogenetic diagnosis in amniocytes.
5. For culturing MSCs (second stage), non‐adhering amniotic fluid cells in the supernatant medium were collected on the fifth day after the primary amniocytes culture and kept until completion of fetal chromosome analysis.
6. The cells then were centrifuged and plated in 5 ml of α‐modified minimum essential medium supplemented with 20% fetal bovine serum (FBS) and 4 ng/ml basic fibroblast growth factor (bFGF) in a 25 cm² flask and incubated at 37°C with 5% humidified CO₂ for MSC culture.

Differentiation assay for MSCs

1. Amniotic fluid‐derived mesenchymal stem cells (AFMSCs) were cultured to confluence and shifted to osteogenic medium (α‐MEM supplemented with 10% FBS, 0.1 µmol/l dexamethason, 10 mmol/l β‐glycerol phosphate, 50 µmol/l ascorbate) and adipogenic medium (α‐MEM supplemented with 10% FBS, 1 µmol/l dexamethasone, 5 µg/ml insulin, 0.5 mmol/l isobutylmethylxanthine and 60 µmol/l indomethacin) for 3 weeks.
2. The differentiation potential for osteogenesis was assessed by the mineralization of calcium accumulation by von Kossa staining.
3. For adipogenic differentiation, intracellular lipid droplets could be observed under the microscope and confirmed by Oil Red O staining.
4. For differentiation of neural cells, AFMSCs were incubated with α‐MEM supplemented with 20% FBS, 1 mmol/l β‐mercaptoethanol, 5 ng/ml bFGF for 24 h, and then treated with serum depletion for 5 h.
5. Immunocytochemical stain with neuron‐specific class III β‐tubulin (TuJ‐1) was used to assess the capacity of neuronal differentiation.

Reference
Moertel CA, Stupca PJ and Dewald GW (1992) Pseudomosaicism, true mosaicism, and maternal cell contamination in amniotic fluid processed with in situ culture and robotic harvesting. Prenat Diagn 12,671–683.